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N Topouzian

Publications and source records attributed to N Topouzian.

6 recordsLinked to original sources
PubMed2001

Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization.

We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of alpha-spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in alpha-spectrin. The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction. Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of alpha- and beta-spectrin at the tetramerization site.

Binding Sites↗
PubMed1996

Peptides with more than one 106-amino acid sequence motif are needed to mimic the structural stability of spectrin.

The primary sequence of human erythrocyte spectrin contains repetitive homologous sequence motifs of approximately 106 amino acids with 22 such motifs in the alpha-subunit and 17 in the beta-subunit. These homologous sequence motifs have been proposed to form domains with a triple-helical bundle type structure (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180; Parry, D. A. D., Dixon, T. W., and Cohen, C. (1992) Biophys. J. 61, 858-867). In this study, we show that these sequence motifs, while they do form compact proteolytically resistant units, are not completely independent. Peptides composed of two or three such motifs in tandem are substantially more stable than peptides composed of a single motif, as measured by proteolysis or by fluorescence or circular dichroism studies of urea or thermal denaturation. Circular dichroism and infrared spectroscopy measurements also indicate that these larger, more stable peptides exhibit greater secondary structure. In these respects, the peptides with tandem sequence motifs are more similar to intact spectrin than the peptide with a single sequence motif. Thus, we conclude that peptides with more than one sequence motif model spectrin more adequately than the peptides with one sequence motif, and that these sequence motifs are not completely independent domains.

Circular Dichroism↗
PubMed1986

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus.

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.

Bifidobacterium↗
PubMed1984

Effect of various metals and calcium metabolism inhibitors on the growth of Bifidobacterium bifidum var. pennsylvanicus.

In view of the facts that the normal intestinal flora exerts beneficial effects and that bifidobacteria are a more important component in the breast-fed than in the bottle-fed infant, factors affecting the growth of the latter microorganisms are of interest. A series of transition and other metals were shown to be growth inhibitors of Bifidobacterium bifidum var. pennsylvanicus. Such inhibition could be reversed fully or partially by 0.5-1.0 mM Fe2+ in the case of Zn2+, Cu2+, Au3+, Pt4+, La3+, Cr3+, Mn2+, Ni2+, and Cd2+, but not with Ag+, Hg2+, and VO2+. In addition, 2-4 mM Ca2+ substantially relieved the inhibitory effects of Zn2+, Mn2+, and La3+, and partially relieved the effects of Cd2+. Mg2+ was ineffective in relieving Zn2+ inhibition, but Ba2+ and Sr2+ could replace Ca2+ to some extent. The calcium metabolism antagonists verapamil, ruthenium red, 2-chloroadenosine, lasalocid, Ca-ionophore A-23187, and calmodulin inhibitors W-5 and W-7 inhibited microbial growth. Inhibition could be relieved fully or partially with 0.5-1 mM Fe2+. Mg2+ relieved the inhibition by lasalocid, Ca-ionophore A-23187, and verapamil, whereas Ca2+ was effective only in the case of Ca-ionophore A-23187. We conclude that calcium and magnesium fluxes play an important role in the physiology of the bifidobacteria and that several metal growth inhibitors interfere with iron metabolism.

Bifidobacterium↗
PubMed1981

The effect of metal chelators and other metabolic inhibitors on the growth of Bifidobacterium bifidus var. Pennsylvanicus.

Bifidobacterium bifidus var. Pennsylvanicus, a microaerophilic anaerobe, was grown in the presence of several potential growth inhibitors with the aim of defining its growth requirements and metabolic peculiarities. The following had no effect on its growth: citrate, serum transferrin, serum albumin, colchicine, fluoro-acetate, malonate, and rotenone. The following substances inhibited the growth: fluoride, azide, arsenite, 2, 4-dinitrophenol, hemin, hemoglobin, lactoferrin, alpha, alpha'-bipyridyl, and 8-hydroxyquinoline. Ferrous iron was able to negate the inhibition achieved by alpha, alpha'-bipyridyl, and 8-hydroxyquinoline. It is concluded that iron, probably in its ferrous state, is an obligatory nutrient for the microorganism, and that iron-porphyrin system(s) may be essential for the metabolism of this organism. Because the microorganisms contained in addition to iron large quantities of Mn, Zn, and Cu, it is likely that these metalloelements are crucial for the normal growth of the organism. Growth inhibition by fluoride indicates that Mg-dependent enzymes may also be present in the microorganism.

2,2'-Dipyridyl↗